Diabetes mellitus is a global epidemic disease and is a metabolic disorder relating to glucose, protein and lipids due to the absolute or relative deficiency of insulin (See Chen Ruijie. Status of research on diabetes drugs. Academic journal of Guangdong College of Pharmacy, 2001, 7(2):131-133). Diabetes mellitus can be divided into type I diabetes mellitus and type II diabetes mellitus (Type 2 diabetes mellitus, T2DM, the same below) according to the pathogenesis thereof 90-95% of all the patients diagnosed with diabetes mellitus suffer from T2DM, and patients are often afflicted with obesity, a deficiency of physical activity. T2DM is most common in the aging population, or among those with family history of diabetes mellitus T2DM. It is also a progressive disease. According to statistical data in 2000, the World Health Organization estimated that there are about 171 million people worldwidely who suffer from diabetes mellitus. In 2005, the U.S. Centers for Disease Control and Prevention. estimated that 20.8 million Americans suffer from diabetes mellitus which is about 7% of the population of the United States. In 2006, the International Diabetes Federation, estimated that the global number of patients suffering from diabetes mellitus is about 246 million (about 5.9% of the totally global population) and indicated that 46% of the patients were 40-59 years old.
T2DM is characterized by the inhibition of the secration of insulin and pancreatic β-cell dysfunction which results in insulin deficiency and hyperglycemia. (See Ferrannini E. Insulin resistance versus insulin deficiency in non-insulin-dependent diabetes mellitus: problems and prospects. Endocr Rev. 1998, 19(4):477-490). T2DM patients typically suffer from a postprandial and fasting hyperglycemia (fasting glucose >125 mg/dL). Observed high blood sugar is the result of pancreatic β-cells failure to secrete enough insulin in the surrounding tissue. (See Weyer C., Bogardus C., Mott D M., et al. The natural history of insulin secretory dysfunction and insulin resistance in the pathogenesis of type 2 diabetes mellitus. J. Clin. Invest. 1999, 104(6): 787-794).
A major risk factor of T2DM is obesity, which is itself very harmful to human health. T2DM often co-exists with other high-risk diseases such as hypertension and dyslipidemia. 60% of T2DM patients are accompanied by microvascular complications, including retinopathy and neuropathy, and also are accompanied by cardiovascular morbidities, such as coronary heart disease, myocardial infarction, shock, and the like. In the U.S., cardiovascular diseases (CVD) is the major cause resulting in mortality, and T2DM is the major risk factor causing macrovascular complications such as an atherosclerosis, myocardial infarction, shock, and peripheral vascular diseases. The risk of death caused by heart diseases with diabetes is 2-4 times higher than that of a non-diabetes person. In addition, nearly 65% of people with diabetes die of heart disease.
In addition to the physical and physiological harm to patients, T2DM causes great economic burden on society. According to statistics, the cost of the treatment of complications associated with diabetes is about $ 22.9 billion; the total cost of the treatment of T2DM and complications thereof is nearly $ 57.1 billion every year in the U.S.
Drugs for the treatment of T2DM have been sought. These include the early oral hypoglycemic drugs of sulfonyl class and biguanide class and the recent insulin sensitizer and α-glucosidase inhibitors, the development of animal insulins and human insulins in a variety of new regimes and formulations, the research of new mechanisms of drug treatment by simply increasing insulin, and new ways acting on the insulin-producing cells. Weight gain is a common side-effect after the administration of oral or injection hypoglycemic agents, which may reduce compliance, and may increase the risk of developing cardiovascular disease. Therefore, developing new types of drugs for the treatment of T2DM which have high safety profiles, good patient compliance and low side-effects is desirable.
As early as 100 years ago, Moore proposed that the duodenum can secrete a “chemical stimulant” stimulating pancreatic secretion. Attempts to inject gut-extract to treat diabetes were undertaken. Subsequently it was discovered that humoral factors derived from intestinal secretion can enhance the function of the pancreas endocrine, and about 50% of insulin secretion induced by intravenous or oral glucose is derived from the stimulus of peptides produced in the gut. Therefore Zunz and Labarre described the concept of “incretin.” Two kinds of incretins have been isolated so far, namely glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1). Both GIP and GLP-1 are secreted by specific intestinal nerve cells when a related nutrient is absorbed. GIP is secreted by the duodenum and proximal jejunal K cells. GLP-1 is synthesized in L cells and mainly exists in the distal small bowel and colon (See Drucker D J. Enhancing incretin action for the treatment of type 2 diabetes. Diabetes Care. 2003, 26(10):2929-2940).
GLP-1 exists in two bio-active forms in blood plasma, namely GLP-1 (7-37) and GLP-1 (7-36). The difference between the two forms resides in one amino acid residue, and their biological effects and in vivo half-life are the same. (See Drucker D J. Enhancing incretin action for the treatment of type 2 diabetes. Diabetes Care. 2003, 26(10):2929-2940).
GLP-1 is usually referred to as GLP-1 (7-37) and GLP-1 (7-36) amide. GIP and GLP-1 are degraded to inactive forms by dipeptidyl peptidase-IV (DPP-IV) quickly after released in the gastrointestinal tract, so that the in vivo half-life of GIP and GLP-1 is very short (in vivo half-life of GIP is about 5-7 minutes, in vivo half-life of GLP-1 is about 2 minutes). (See Drucker D J. Enhancing incretin action for the treatment of type 2 diabetes. Diabetes Care. 2003, 26(10):2929-2940). Researches show that most of the degradation process occurs when the GIP and GLP-1 enter into the blood vessels containing DPP-IV, and a small amount of GLP-1 and GIP which has not been degraded will enter into the pancreas and associate with binding sites to stimulate insulin release from β-cells. Different from the mechanism of sulfonylurea to directly promote functional β-cells to release insulin, most of the effects of incretin are glucose-dependent. In addition, some in vitro tests on animals and humans have shown that GLP-1 also functions to suppress α-cell and reduce glucagon hypersecretion.
Although plasma GIP levels in patients with T2DM are normal, when the function of incretin declines significantly, the GLP-1 levels in patients with T2DM decline. Thus, drugs based on GLP-1 contribute more to treatment of T2DM. Although the levels of both GLP-1 (7-37) and GLP-1 (7-36) amide will increase in several minutes after a meal, and the content of GLP-1 (7-36) amide is more, so the GLP-1 secretion might have been greatly increased by the double effect of endocrine and transmission of neural signal before the digested food enters the small intestine and colon. The plasma level of GLP-1 under a fasting state is very low (about 5-10 pmol/L), and is increased rapidly after eating (up to 15-50 pmol/L). Under the double function of DPP-IV and renal clearance, the level in vivo of GLP-1 in circulation is decreased rapidly. Other enzymes such as human neutral endopeptidase 24•11 may also play a vital role in inactivating clearacne of GLP-1. Because the second amino acid residue of GLP-1 is alanine, which is a good substrate of DPP-IV, GLP-1 is easily degraded into inactive peptide fragments. In fact, the DPP-IV in vivo is postulated as the key reason for loss of the activity of the incretin. Experiments show that GLP-1 levels in mice, in which DPP-IV gene has been silenced, is higher than in normal mice. Significantly, insulin secretion is increased, too. Just because the presence of DPP-IV, the content in vivo (except in plasma) of the nondegradaded and biologically active GLP-1 is only 10-20% of the total content of GLP-1 in plasma. (See Deacon C F, Nauck M A, Toft-Nielsen M, et al. Both subcutaneously and intravenously administered, glucagon-like peptide 1 is rapidly degraded from the NH2-terminus in type 2-diabetic patients and in healthy subjects. (See Diabetes. 1995, 44(9): 1126-1131).
GLP-1 and GIP play their respective roles through binding to different G-protein-coupled receptors (GPCRs). Most of GIP receptors are expressed by pancreatic β-cells, and a minor part of GIP receptors are expressed by adipose tissue and the central nervous system. In contrast, GLP-1 receptors are mainly expressed in the pancreatic α- and β-cells and peripheral tissues including the central and peripheral nervous systems, brain, kidney, lung and gastrointestinal tract and the like. The activation of two incretins in β-cells will result in the rapid increase of the level of cAMP and intracellular calcium, thereby rleading to their extracellular secretion in a glucose-dependent manner. The sustained signal transmission from incretin receptors is associated with protein kinase A, resulting in gene transcription, increasing insulin biosynthesis and stimulating β-cell proliferation. (See Gallwitz B. Glucagon-like peptide-1-based therapies for the treatment of type 2 diabetes mellitus. Treat Endocrinol. 2005, 4(6):361-370). The activation of GLP-1 receptor and GIP receptor can also inhibit the apoptosis of pancreatic β-cells of rodent and human, while increasing their survival (See Li Y, Hansotia T, Yusta B, et al. Glucagon-like peptide-1 receptor signaling modulates beta cell apoptosis. J Biol Chem. 2003, 278(1): 471-478). Consistent with the expression of GLP-1 receptor, GLP-1 can also inhibit glucagon secretion, gastric emptying and food intake, and enhance the degradation of glucose through the neural mechanism. It shall be noted that, as with other insulin secretion mechanisms, the role of GLP-1 to control the level of glucose is glucagon-dependent and the counter-regulatory release of glucagon caused by low blood sugar is fully retained even at the pharmacological level of GLP-1.
The important physiological role of endogenous GLP-1 and GIP in glucose homeostasis has been studied in-depth through using receptor antagonists or gene knockout mice. Acute antagonism of GLP-1 or GIP reduces insulin secretion in vivo of rodents and increases plasma glucose content. Similarly, the mutant mice, in which GIP or GLP-1 receptor is inactivated, also experience defective glucose-stimulated insulin secretion and damaged glucose tolerance. GLP-1 also has a function of regulating fasting blood glucose, because the acute antagonists or damage on the GLP-1 gene will cause the increase of fasting glucose level of rodents. At the same time, GLP-1 is the basis of glucose control in human bodies, and studies on the antagonist of Exendin (9-39) have shown that the destruction of GLP-1 function will result in defective glucose-stimulated insulin secretion, decreased glucose clearance rate, increased glucagon levels and accelerated gastric emptying. The physiological roles of GLP-1 (see Deacon C F. Therapeutic strategies based on glucagon-like peptide 1. Diabetes. 2004, 53(9):2181-2189) comprise: (1) helping to organize glucose absorption, mediate glucose-dependent insulin secretion; (2) inhibiting postprandial glucagon secretion, reducing hepatic glucose release; (3) regulating gastric emptying, preventing excessive circulating of glucose when the food is absorbed in the intestine; and (4) inhibiting food intake (such as appetite). Also, animal studies also showed a physiological role for stabilizing the number of pancreatic β-cells in vivo.
Due to the beneficial effects of GLP-1 and GIP in controlling blood sugar and many other aspects, especially their characteristics of not producing hypoglycemia and delaying gastric emptying to control weight, the compounds attract the interest of many scientists. Further studies of based on GLP-1 and GIP for the treatment of T2DM have been pursued. It is well known that T2DM patients lack or lose the incretin effect. One reason is that incretin effect of GIP in vivo in the T2DM patient is significantly reduced. Meanwhile, the level of GLP-1 in vivo in T2DM patients is very low, and the level of GLP-1 caused by dietary stimuli is significantly reduced. (See Toft-Nielsen M B, Damholt M B, Madsbad S, et al. Determinants of the impaired secretion of glucagon-like peptide-1 in type 2 diabetic patients. J Clin Endocrinol Metab. 2001, 86(8):3717-3723). Because the role of GLP-1 in vivo in patients with T2DM has been partially reserved, GLP-1 synergist is one of the research directions of the drugs designed to enhance the incretin effect in T2DM patients.
GLP-1 analogues, may act similarly to endogenous GLP, by inhibiting the release of glucagon and stimulate insulin secretion both in vivo in a glucose-dependent manner and thus its role for lowering blood glucose exhibit a self-limitation, which generally does not cause hypoglycemia in large doses. Some literature reports that GLP-1 can reduce blood sugar to a level below normal, and this effect is transient and considered a natural result of GLP-1 promoted insulin secretion. GLP-1 can temporarily reduce blood sugar to a level below normal level but does not cause serious and persistent hypoglycemia. Besides directly reducing blood glucose, GLP-1 can also reduce the quantity of food intake, which has been verified in rodents and humans. The level of blood glucose, therefore, can be controlled by reducing body weight indirectly. GLP-1 also has the potential role of inhibiting the secretion of gastrin and gastric acid stimulated by eating, and these functions show that GLP-1 may also have a role in the prevention of peptic ulcer. Mechanisms of action for GLP-1 make it an ideal drug for the treatment of patients with type 2 diabetes, but also the drug for the treatment of patients with obesity diabetes. GLP-1 can enhance the satiety of the patients, reduce food intake and maintain body weight or lose weight. Several studies suggest that GLP-1 can prevent the conversion from impaired glucose tolerance to diabetes, and some literature reports that the GLP-1 class of compounds has direct effect on the growth and proliferation of pancreatic β-cells in experimental animals. It was found by some experiments that GLP-1 can promote the differentiation from pancreatic stem cells to functional β-cells. These results suggest that GLP-1 has the function of protecting pancreatic islet and delaying the progression of diabetes, and can maintain the morphologies and functions of β-cells, while reduce the apoptosis of β-cells. Because some oral drugs and exogenous insulins can not inhibit or reduce the exorbitant glucagon secretion in patients with T2DM, GLP-1 analogues can affect glucagon hypersecretion through directly inhibiting glucagon release or inhibition of glucagon resulted from promoting insulin secretion. The postprandial hyperglycemia can be reduced effectively through these two mechanisms. Meanwhile, the maintaining of the function of β-cells may also play a role in controlling the long-term postprandial hyperglycemia.
GLP-1 analogues are administered through subcutaneous injection, which doesn't require calculation of the amount of carbohydrates to estimate the optimal drug dosage, and does not require self-monitoring the blood glucose. As a result, these kinds of drugs are easier for patient compliance than self-administered insulin.
A variety of effects of natural GLP-1 have been confirmed, which bring new hope for the treatment of T2DM. The natural human GLP-1 peptide is, however, very unstable and can be degraded by dipeptidyl peptidase IV (DPP-IV). Moreover, its half-life is only about 2 minutes. When using natural GLP-1 to lower blood sugar, continuous intravenous infusion or continuous subcutaneous injection is needed, resulting in its poor clinical feasibility. Faced with this situation, researchers continue to explore methods to extend the action time of GLP-1. Therefore, there is a need for the development of long-acting GLP-1 analogues or derivatives thereof.
Exenatide is a synthetic Exendin-4, which is developed by the Eli Lilly Company and Amylin Company, with the trade name Byetta®. Exenatide has been approved for the treatment of T2DM by FDA and EMEA. It has 50% homology with mammalian GLP-1 in sequence and has a similar affinity site of the receptor with GLP-1. (See Drucker D J, Nauck M A. The incretin system: glucagon-like peptide-1 receptor agonists and dipeptidyl peptidase-4 inhibitors in type 2 diabetes. Lancet. 2006, 368(9548):1696-1705). It is encoded by a lizard-specific gene. Compared with GLP-1, the second residue, alanine, in GLP-1 is replaced with glycine in Exenatide, which effectively inhibits the enzymolysis of DPP-IV enzyme, and its half-life in vivo is about 60-90 minutes. (See Kolterman O G, Kim D D, Shen L, et al. Pharmacokinetics, pharmacodynamics, and safety of exenatide in patients with type 2 diabetes mellitus. Am Health Syst Pharm. 2005, 62(2): 173-181). The in vivo concentration of Exenatide after a single subcutaneous injection is increased persistently and can arrive to the maximum plasma concentration after 2 h or so, which can be maintained for 4-6 hours. (See Nielsen L L, Baron A D. Pharmacology of exenatide (synthetic exendin-4) for the treatment of type 2 diabetes. Curr Opin Investig Drugs. 2003, 4(4):401-05). It should be noted that the metabolism of Exenatide does not occur in the liver, but is degraded mainly by protein protease after filtered by renal glomeruli.
Exenatide has special glucose-regulating activities, including glucose-dependent enhance of insulin secretion, glucose-dependent inhibition of wrong excessive glucagon secretion, slowing gastric emptying and decreasing food intake and the like. Studies in vitro and in vivo in the models of diabetes found that Exenatide also has the effects of storing the first stage (first-phase) insulin secretion, promoting the proliferation of β-cell and promoting the regeneration of insulin from its precursor cell.
In order to achieve better control of blood glucose, injections twice a day of Exenatide are needed. This is a major inconvenience to patients. Furthermore, Exenatide has unfortunate side effects including mild to moderate nausea (about 40% of patients will have this reaction), diarrhea and vomiting (less than 15% of patients have both reactions). In addition, about 50% of Exenatide-treated patients can generate antibodies, although these antibodies do not affect the efficacy or lead to other clinical effects. Recently it is found that six patients suffered hemorrhage or symptoms of necrotizing pancreatitis after taking Byetta®.
CJC-1131 is a GLP-1 analogue with peptidase resistance developed by ConjuChem Biotechnologies Inc., in which the alanine residue in the second position of GLP-1 is replaced with D-Ala to enhance resistance of DPP-IV enzymolysis. The structure contains an active reactive linker that can bind to serum albuminutes through a covalent, non-reversible manner. (See Kim J G, Baggio L L, Bridon D P, et al. Development and characterization of a glucagon-like peptide-1 albuminutes conjugate: the ability to activate the glucagon-like peptide 1 receptor in vivo. Diabetes 2003, 52(3):751-759). The GLP-1-serum albuminutes complex retains the activity of GLP-1, while increasing its stability to DPP-IV enzymolysis, thereby extending in vivo action. Its half-life in plasma is about 20 days.
A study has found that the Ki was approximate 12 nM (the Ki of GLP-1 is 5.2 nM) when CJC-1131-serum albuminutes complex is bound to Chinese hamster ovary cell transfected with recombinant human pancreatic GLP-1 receptor. Meanwhile the EC50 of the complex activating cAMP is 11-13 nM, wherein the EC50 is similar to GLP-1's EC50. Existing literature reports show that this complex can reduce postprandial blood glucose level of the mice whose blood sugar is normal or high, and tests show that this activity of CJC-1131 acts on a certain functional receptor of GLP-1. Meanwhile in mice, CJC-1131 also has an effect on slowing gastric emptying and inhibiting food intake and the like.
Part of a phase II clinical trial of CJC-1131 has been completed. In September 2005, ConjuChem concluded that CJC-1131 may not be suitable for chronic dosing regimens after analysis of test results and suspended further clinical study
Albugon (albumin-GLP-1) is a long-acting drug for the treatment of T2DM developed by GlaxoSmithKline authorized by Human Genome Sciences Inc., which is a fusion protein of GLP-1 (with mutations increasing the resistance to DDP-IV) and albumin. Its half-life in monkeys is 3 days. The basic idea of the development thereof is to couple the recombinant GLP-1 and serum albuminutes to form a complex, thereby its in vivo half-life is significantly increased. The administration of Albugon effectively reduces blood glucose level of mice, increases insulin secretion, slows gastric emptying and reduces food intake etc. (See Baggio L L, Huang Q, Brown T J, et al. A Recombinant Human Glucagon-Like Peptide (GLP)-1-Albuminutes Protein (Albugon) Mimics Peptidergic Activation of GLP-1Receptor-Dependent Pathways Coupled With Satiety, Gastrointestinal Motility, and Glucose Homeostasis. Diabetes 2004, 53(9):2492-2500). Currently Albugon is in phase III clinical trials.
WO9808871 discloses a GLP-1 derivative which is obtained through the modification on GLP-1(7-37) with fatty acid. The half life in vivo of GLP-1 is significantly enhanced. WO9943705 discloses a derivative of GLP-1, which is chemically modified at the N-terminus, but some literature reports that modification of the amino acids on the N-terminal will significantly decrease the activity of the entire GLP-1 derivative. (See J. Med. Chem. 2000, 43, 1664 1669). In addition, CN200680006362, CN200680006474, WO2007113205, CN200480004658, CN200810152147 and WO2006097538 etc also disclose a series of GLP-1 analogues or derivatives thereof produced by chemical modification or amino acid substitution, in which the most representative one is liraglutide developed by Novo Nordisk, the phase III clinical trial of which has been finished. Liraglutide is a derivative of GLP-1, whose structure contains a GLP-1 analogue of which the sequence is 97% homologous with human GLP-1, and this GLP-1 analogue is linked with palmitic acid covalently to form Liraglutide, wherein the palmitic acid of the structure of Liraglutide is linked to serum albuminutes non-covalently, and this structural characteristic affects a slower release from the injection site without changing the activity of GLP-1 thereby extending its in vivo half life Meanwhile, the palmitic acid in the structure will form a certain steric hindrance to prevent the degradation by DPP-IV and to reduce renal clearance. Because of the characteristics described above, the half-life of Liraglutide in the human body administered by subcutaneous injection is about 10-14 hours. In theory, it can be administered once on day and the daily dose is 0.6-1.8 mg. On Apr. 23, 2009, Novo Nordisk announced that Committee for Medicinal products for Human Use (CHMP) under the EMEA gave a positive evaluation on Liraglutide and recommended approval of its listing. Novo Nordisk hopes that European Commission would approve its application of listing within two months.